Virtual reality (VR), a computer-generated environment which simulates sensory experience in the real world, has been used to study neural substrates of behavior in primates and humans for many years and has only recently become an important technology in rodent neuroscience. Towards imaging hippocampal neural circuit activity during spatial behavior, we built a VR set-up around a two-photon microscope and trained mice in behavioral tasks in VR. In this environment, mice with their heads fixed above an air-supported spherical treadmill were allowed to run freely. Computer-generated VR scenes were rendered on a wide LCD monitor to provide visual feedback in response to running. Mice trained in a virtual linear track learned to exhibit spontaneous alteration of running and standing still, which allowed us to study behavioral-state dependent changes of hippocampal neuronal ensemble dynamics with calcium imaging. To test whether mice can discriminate a particular "place" in VR, we also established a new virtual spatial recognition task. In this task, mice start running from one end of a visual cue-rich virtual linear track to the other end. To receive water reward, they need to recognize location of a reward zone in the middle of the track and stay there for a period of delay. Rapid behavioral plasticity can be also induced by moving the position of the reward zone and making the mice relearn the new location. Overall, our reserach demonstrates that mice can develop robust spatial behavior in VR. These behavioral paradigms will thus facilitate studies on cellular and circuit mechanisms underlying spatial navigation and memory as well as their dysfunction associated with developmental and psychiatric disorders.